Endothelial Extracellular Vesicles: From Keepers of Health to Messengers of Disease.

Endothelium has a rich vesicular network that allows the exchange of macromolecules between blood and parenchymal cells. This feature of endothelial cells, along with their polarized secretory machinery, makes them the second major contributor, after platelets, to the particulate secretome in circulation. Extracellular vesicles (EVs) produced by the endothelial cells mirror the remarkable molecular heterogeneity of their parent cells. Cargo molecules carried by EVs were shown to contribute to the physiological functions of endothelium and may support the plasticity and adaptation of endothelial cells in a paracrine manner. Endothelium-derived vesicles can also contribute to the pathogenesis of cardiovascular disease or can serve as prognostic or diagnostic biomarkers. Finally, endothelium-derived EVs can be used as therapeutic tools to target endothelium for drug delivery or target stromal cells via the endothelial cells. In this review we revisit the recent evidence on the heterogeneity and plasticity of endothelial cells and their EVs. We discuss the role of endothelial EVs in the maintenance of vascular homeostasis along with their contributions to endothelial adaptation and dysfunction. Finally, we evaluate the potential of endothelial EVs as disease biomarkers and their leverage as therapeutic tools.

Endothelial-to-Mesenchymal Transition in Human Adipose Tissue Vasculature Alters the Particulate Secretome and Induces Endothelial Dysfunction.

OBJECTIVE: Endothelial cells (EC) in obese adipose tissue (AT) are exposed to a chronic proinflammatory environment that may induce a mesenchymal-like phenotype and altered function. The objective of this study was to establish whether endothelial-to-mesenchymal transition (EndoMT) is present in human AT in obesity and to investigate the effect of such transition on endothelial function and the endothelial particulate secretome represented by extracellular vesicles (EV). Approach and Results: We identified EndoMT in obese human AT depots by immunohistochemical co-localization of CD31 or vWF and α-SMA (alpha-smooth muscle actin). We showed that AT EC exposed in vitro to TGF-ß (tumor growth factor-ß), TNF-α (tumor necrosis factor-α), and IFN-γ (interferon-γ) undergo EndoMT with progressive loss of endothelial markers. The phenotypic change results in failure to maintain a tight barrier in culture, increased migration, and reduced angiogenesis. EndoMT also reduced mitochondrial oxidative phosphorylation and glycolytic capacity of EC. EVs produced by EC that underwent EndoMT dramatically reduced angiogenic capacity of the recipient naïve ECs without affecting their migration or proliferation. Proteomic analysis of EV produced by EC in the proinflammatory conditions showed presence of several pro-inflammatory and immune proteins along with an enrichment in angiogenic receptors. CONCLUSIONS: We demonstrated the presence of EndoMT in human AT in obesity. EndoMT in vitro resulted in production of EV that transferred some of the functional and metabolic features to recipient naïve EC. This result suggests that functional and molecular features of EC that underwent EndoMT in vivo can be disseminated in a paracrine or endocrine fashion and may induce endothelial dysfunction in distant vascular beds.

Countering the Modern Metabolic Disease Rampage With Ancestral Endocannabinoid System Alignment.

When primitive vertebrates evolved from ancestral members of the animal kingdom and acquired complex locomotive and neurological toolsets, a constant supply of energy became necessary for their continued survival. To help fulfill this need, the endocannabinoid (eCB) system transformed drastically with the addition of the cannabinoid-1 receptor (CB1R) to its gene repertoire. This established an eCB/CB1R signaling mechanism responsible for governing the whole organism's energy balance, with its activation triggering a shift toward energy intake and storage in the brain and the peripheral organs (i.e., liver and adipose). Although this function was of primal importance for humans during their pre-historic existence as hunter-gatherers, it became expendable following the successive lifestyle shifts of the Agricultural and Industrial Revolutions. Modernization of the world has further increased food availability and decreased energy expenditure, thus shifting the eCB/CB1R system into a state of hyperactive deregulated signaling that contributes to the 21st century metabolic disease pandemic. Studies from the literature supporting this perspective come from a variety of disciplines, including biochemistry, human medicine, evolutionary/comparative biology, anthropology, and developmental biology. Consideration of both biological and cultural evolution justifies the design of improved pharmacological treatments for obesity and Type 2 diabetes (T2D) that focus on peripheral CB1R antagonism. Blockade of peripheral CB1Rs, which universally promote energy conservation across the vertebrate lineage, represents an evolutionary medicine strategy for clinical management of present-day metabolic disorders.

Activation of the 12/15 lipoxygenase pathway accompanies metabolic decline in db/db pre-diabetic mice.

The 12-lipoxygenase (12LO) pathway is a promising target to reduce islet dysfunction, adipose tissue (AT) inflammation and insulin resistance. Optimal pre-clinical models for the investigation of selective12LO inhibitors in this context have not yet been identified. The objective of this study was to characterize the time course of 12LO isoform expression and metabolite production in pancreatic islets and AT of C57BLKS/J-db/db obese diabetic mouse in a pre-diabetic state in order to establish a suitable therapeutic window for intervention with selective lipoxygenase inhibitors. Mice have 2 major 12LO isoforms -the leukocyte type (12/15LO) and the platelet type (p12LO) and both are expressed in islets and AT. We found a sharp increase in protein expression of 12/15LO in the pancreatic islets of 10-week old db-/- mice compared to 8- week old counterparts. Immunohistochemistry showed that the increase in islet 12/15LO parallels a decline in islet number. Analysis of 12- and 15-hydroperoxytetraeicosanoid acids (HETE)s showed a 2-3 fold increase especially in 12(S)-HETE that mirrored the increase in 12/15LO expression in islets. Analysis of AT and stromal vascular fraction (SVF) showed a significant increase of platelet 12LO gene expression along with 12- and 15- HETEs. The data demonstrate that the db/db mouse is a suitable model for investigation of 12/15LO inhibitors in the development of inflammatory mediated type 2 diabetes, with a narrow window of therapeutic intervention prior to 8 weeks of age.

Atheroprotection through SYK inhibition fails in established disease when local macrophage proliferation dominates lesion progression.

Macrophages in the arterial intima sustain chronic inflammation during atherogenesis. Under hypercholesterolemic conditions murine Ly6C(high) monocytes surge in the blood and spleen, infiltrate nascent atherosclerotic plaques, and differentiate into macrophages that proliferate locally as disease progresses. Spleen tyrosine kinase (SYK) may participate in downstream signaling of various receptors that mediate these processes. We tested the effect of the SYK inhibitor fostamatinib on hypercholesterolemia-associated myelopoiesis and plaque formation in Apoe(-/-) mice during early and established atherosclerosis. Mice consuming a high cholesterol diet supplemented with fostamatinib for 8 weeks developed less atherosclerosis. Histologic and flow cytometric analysis of aortic tissue showed that fostamatinib reduced the content of Ly6C(high) monocytes and macrophages. SYK inhibition limited Ly6C(high) monocytosis through interference with GM-CSF/IL-3 stimulated myelopoiesis, attenuated cell adhesion to the intimal surface, and blocked M-CSF stimulated monocyte to macrophage differentiation. In Apoe(-/-) mice with established atherosclerosis, however, fostamatinib treatment did not limit macrophage accumulation or lesion progression despite a significant reduction in blood monocyte counts, as lesional macrophages continued to proliferate. Thus, inhibition of hypercholesterolemia-associated monocytosis, monocyte infiltration, and differentiation by SYK antagonism attenuates early atherogenesis but not established disease when local macrophage proliferation dominates lesion progression.

A novel CX3CR1 antagonist eluting stent reduces stenosis by targeting inflammation.

We evaluated the therapeutic efficacy of a novel drug eluting stent (DES) inhibiting inflammation and smooth muscle cell (SMC) proliferation. We identified CX3CR1 as a targetable receptor for prevention of monocyte adhesion and inflammation and in-stent neointimal hyperplasia without interfering with stent re-endothelization. Efficacy of AZ12201182 (AZ1220), a CX3CR1 antagonist was evaluated in inhibition of monocyte attachment in vitro. A prototype AZ1220 eluting PLGA-based polymer coated stent developed with an optimal elution profile and dose of 1 µM/stent was tested over 4 weeks in a porcine model of coronary artery stenting. Polymer coated stents without AZ1220 and bare metal stents were used as controls. AZ1220 inhibited monocyte attachment to CX3CL1 in a dose dependent manner. AZ1220 eluted from polymer coated stents in an ex vivo flow system retained bioactivity in inhibiting monocyte attachment to CX3CL1. At 4 weeks following deployment, AZ1220 eluting stents significantly reduced (∼60%) in-stent stenosis compared to both bare metal and polymer only coated stents and markedly reduced peri-stent inflammation and monocyte/macrophage accumulation without affecting re-endothelization. Anti-CX3CR1 drug eluting stents potently inhibited in-stent stenosis and may offer an alternative to mTOR targeting by current DES, specifically inhibiting polymer-induced inflammatory response and SMC proliferation, while retaining an equivalent re-endothelization response to bare metal stents.

Fractalkine has anti-apoptotic and proliferative effects on human vascular smooth muscle cells via epidermal growth factor receptor signalling.

AIMS: Fractalkine (CX3CL1) is a membrane-bound chemokine that signals through the G protein-coupled receptor CX3CR1 that is implicated in the development of atherosclerosis. We have previously reported that CX3CR1 is expressed by primary human coronary artery smooth muscle cells (CASMC), where it mediates chemotaxis towards CX3CL1. We sought to determine the effect of CX3CL1 on CASMC survival and proliferation and elucidate the signalling mechanisms involved. METHODS AND RESULTS: CX3CL1 significantly reduces staurosporine-induced apoptosis of CASMC, as quantified by caspase 3 immunostaining and Annexin-V flow cytometry. Furthermore, CX3CL1 is a potent mitogen for primary CASMC and induces phosphorylation of extracellular signal-regulated kinase (ERK) and Akt, measured by western blotting. Inhibition of either ERK or phosphoinositide 3-kinase (PI3K) signalling abrogates proliferation, while only PI3K signalling is involved in the anti-apoptotic effects of CX3CL1. We describe a novel and specific small molecule antagonist of CX3CR1 (AZ12201182) which abrogates the mitogenic and anti-apoptotic effects of CX3CL1 on CASMC. Pharmacological inhibition of the epidermal growth factor receptor (EGFR) blocks CASMC survival and DNA synthesis, indicating a previously undocumented role for EGFR signalling in response to CX3CL1 involving release of a soluble EGFR ligand. Specifically, CX3CL1 induces shedding of epiregulin and increases epiregulin mRNA expression 20-fold within 2 h. Finally, antibody neutralization of epiregulin abrogates the mitogenic effect of CX3CL1. CONCLUSION: We have demonstrated two novel and important functions of CX3CL1 on primary human SMCs: anti-apoptosis and proliferation, both mediated via epiregulin-induced EGFR signalling. Our data have important implications in vascular pathologies including atherosclerosis, restenosis, and transplant accelerated arteriosclerosis, where the balance of SMC proliferation and apoptosis critically determines both plaque stability and vessel stenosis.

The potential link between atherosclerosis and the 5-lipoxygenase pathway: investigational agents with new implications for the cardiovascular field.

The 5-lipoxygenase pathway is responsible for the production of leukotrienes--inflammatory lipid mediators that have a role in innate immunity, but that can also have pathological effects in inflammatory diseases. Recently, a potential link between leukotriene production and atherosclerosis has been proposed. The expression of leukotriene biosynthetic enzymes and leukotriene receptors has been identified in coronary and carotid atherosclerotic plaques, and the levels of biosynthetic enzymes have been correlated with the clinical symptoms of unstable plaques. Genetic variants in 5-lipoxygenase pathway genes have also been associated with a relative risk of developing myocardial infarction and stroke. On the basis of these discoveries, antileukotriene compounds are now being evaluated for the treatment of cardiovascular disease. Several tool compounds have been shown to limit the progression of lesion development in preclinical models of atherosclerosis, and three compounds, including two drugs previously developed for asthma, are undergoing clinical trials in patients with acute coronary syndromes.

Effect of broad-spectrum matrix metalloproteinase inhibition on atherosclerotic plaque stability.

OBJECTIVE: Matrix metalloproteinases (MMPs) form a large family of enzymes that collectively can degrade all components of the extracellular matrix, and there is widespread interest in developing MMP inhibitors for the prevention of atherosclerotic plaque rupture. We have therefore investigated the effects of a broad-spectrum MMP inhibitor, RS-130830, on plaque development and stability. This compound inhibits a wide range of MMPs at concentrations below 20 nmol/L. METHODS: Apolipoprotein E knockout mice were fed a Western diet. Dietary administration of RS-130830 commenced at the same time as fat-feeding and continued for 8, 12, 26 or 36 weeks. To investigate the effect of RS-130830 on established plaques, mice were fed high-fat diet for 16 weeks before initiation of drug treatment and were terminated 20 weeks after this. RESULTS: Broad-spectrum MMP inhibition was associated with a significant increase in plaque area, but there was no change in the incidence of plaque rupture. There were unfavourable changes in phenotypic characteristics associated with plaque instability, such as an increased lipid content and decreased collagen content. CONCLUSIONS: These data suggest that broad-spectrum MMP inhibition RS-130830 does not have a beneficial effect on atherosclerosis in the apolipoprotein E knockout mouse model, and indicate that more selective compounds would be preferable.

Plasma interleukin (IL)-18 concentrations is elevated in patients with previous myocardial infarction and related to severity of coronary atherosclerosis independently of C-reactive protein and IL-6.

AIM: Interleukin-18 (IL-18) is a pro-inflammatory cytokine with a central role in the inflammatory cascade. In the present study, we investigated whether patients with precocious myocardial infarction have higher plasma IL-18 concentrations than matched controls. Furthermore, the relationships between plasma IL-18 concentrations and coronary atherosclerosis, C-reactive protein (CRP), interleukin-6 (IL-6) and traditional cardiovascular risk factors were examined. METHODS AND RESULTS: Three hundred eighty-seven unselected survivors of a first myocardial infarction aged less than 60 years and 387 sex and age matched controls were enrolled in the study. A subset of patients (n=236) was evaluated by quantitative coronary angiography. Postinfarction patients had significantly higher mean level of plasma IL-18 than controls (309.6+/-138.6 versus 285.4+/-115.7pg IL-18/mL). Furthermore, plasma IL-18 concentration was significantly associated with coronary plaque area (r=0.17, p=0.009). This relationship remained in a partial correlation analysis adjusting for CRP (r=0.15, p=0.02), for IL-6 (r=0.15, p=0.02) and for both CRP and IL-6 (r=0.15, p=0.02). In addition, IL-18 levels were significantly associated with other cardiovascular risk factors, namely age, low density lipoprotein (LDL) cholesterol, triglycerides, high density lipoprotein (HDL) cholesterol, insulin, proinsulin, body mass index (BMI), systolic and diastolic blood pressure. CONCLUSION: The present work provides evidence that plasma IL-18 is increased in postinfarction patients and is associated with coronary atherosclerosis.

Hypoxia increases LDL oxidation and expression of 15-lipoxygenase-2 in human macrophages.

OBJECTIVE: Macrophage-mediated oxidation of low-density lipoprotein (LDL) by enzymes, such as the lipoxygenases, is considered of major importance for the formation of oxidized LDL during atherogenesis. Macrophages have been identified in hypoxic areas in atherosclerotic plaques. METHODS AND RESULTS: To investigate the role of hypoxia in macrophage-mediated LDL oxidation, we incubated human monocyte-derived macrophages with LDL under normoxic (21% O2) or hypoxic (0% O2) conditions. The results showed that hypoxic macrophages oxidized LDL to a significantly higher extent than normoxic cells. Interestingly, the mRNA and protein expression of 15-lipoxygenase-2 (15-LOX-2) as well as the activity of this enzyme are elevated in macrophages incubated at hypoxia. Both the unspliced 15-LOX-2 and the spliced variant 15-LOX-2sv-a are found in macrophages. In addition, 15-LOX-2 was identified in carotid plaques in some macrophage-rich areas but was only expressed at low levels in nondiseased arteries. CONCLUSIONS: In summary, these observations show for the first time that 15-LOX-2 is expressed in hypoxic macrophages and in atherosclerotic plaques and suggest that 15-LOX-2 may be one of the factors involved in macrophage-mediated LDL oxidation at hypoxia.

Differences in matrix metalloproteinase-1 and matrix metalloproteinase-12 transcript levels among carotid atherosclerotic plaques with different histopathological characteristics.

BACKGROUND AND PURPOSE: Previous studies have shown that atherosclerotic lesions express a number of matrix metalloproteinases (MMPs). Here we investigated whether transcript levels of MMP-1, -3, -7, -9, and -12 in carotid atherosclerotic plaques were correlated with histological features and clinical manifestations. METHODS: Atherosclerotic plaques (n=50) removed from patients undergoing carotid endarterectomy were classified histologically using a system proposed by Virmani et al, and MMP-1, -3, -7, -9, and -12 transcript levels in these tissues were quantified by real-time reverse-transcriptase polymerase chain reaction. RESULTS: Compared to plaques with a thick fibrous cap, those with a thin cap had a 7.8-fold higher MMP-1 transcript level (P=0.006). MMP-3, -7, and -12 were 1.5-fold, 1.8-fold, and 2.1-fold, respectively, higher in thin cap plaques, but the differences did not reach statistical significance. MMP-12 transcript levels were significantly increased in ruptured plaques compared with lesions without cap disruption (P=0.001). MMP-9 transcript levels were similar among the different types of lesion. MMP-1 and -12 transcript levels were significantly higher in plaques from patients with amaurosis fugax, than in those from asymptomatic patients (P=0.029 and P=0.008 for MMP-1 and MMP-12, respectively), than in those from patients with stroke (P=0.027 and P=0.001, respectively), and than in those from patients with transient ischemic attack (P=0.046 and P=0.008, respectively). CONCLUSIONS: These data support a role of MMP-1 and -12 in determining atherosclerotic plaque stability.

A major locus conferring susceptibility to infection by Streptococcus pneumoniae in mice.

We have studied the genetics of susceptibility to infection by Streptococcus pneumoniae in mice. Linkage analysis of the F(2) generation from a cross between resistant BALB/cO1aHsd and susceptible CBA/CaO1aHsd strains allowed us to map a major locus controlling the development of bacteremia and survival after intranasal infection.

Rupture of the atherosclerotic plaque: does a good animal model exist?

By its very nature, rupture of the atherosclerotic plaque is difficult to study directly in humans. A good animal model would help us not only to understand how rupture occurs but also to design and test treatments to prevent it from happening. However, several difficulties surround existing models of plaque rupture, including the need for radical interventions to produce the rupture, lack of direct evidence of rupture per se, and absence of convincing evidence of platelet- and fibrin-rich thrombus at the rupture site. At the present time, attention should therefore focus on the processes of plaque breakdown and thrombus formation in humans, whereas the use of animal models should probably be reserved for studying the function of particular genes and for investigating isolated features of plaques, such as the relationship between cap thickness and plaque stability.

MBL genotype and risk of invasive pneumococcal disease: a case-control study.

BACKGROUND: Streptococcus pneumoniae is a major cause of morbidity and mortality in developed and developing countries. No common genetic determinants of susceptibility have been defined. Mannose-binding lectin (MBL) is a key mediator of innate host immunity that activates the complement pathway and directly opsonises some infectious pathogens. Mutations in three codons in the MBL gene have been identified, and individuals homozygous for a mutant genotype have very little or no serum MBL. We did a case-control study in the UK to assess whether these mutant genotypes were associated with invasive pneumococcal disease. METHODS: The frequencies of genotypes defined by the three mutations in codons 52, 54, and 57, and a functional promoter polymorphism at -221, were compared in a two-stage study of 337 patients with invasive pneumococcal disease and 1032 controls. All individuals were recruited from an ethnically homogeneous white population in Oxfordshire, UK. Patients had S pneumoniae isolated from a normally sterile site. FINDINGS: In our initial set of participants, 28 (12%) of 229 patients and 18 (5%) of 353 controls were homozygotes for MBL codon variants (odds ratio 2.59 [95% CI 1.39-4.83], p=0.002). Neither heterozygosity for these codon variants nor the promoter polymorphism was associated with susceptibility. In a confirmatory study, 11 (10%) of 108 patients were MBL homozygotes compared with 36 (5%) of 679 controls (p=0.046). INTERPRETATION: Homozygotes for MBL codon variants, who represent about 5% of north Europeans and north Americans and larger proportions of populations in many developing countries, could be at substantially increased risk of invasive pneumococcal disease.

Crystal structure of human MMP9 in complex with a reverse hydroxamate inhibitor.

Matrix metalloproteinases (MMPs) and their inhibitors are important in connective tissue re-modelling in diseases of the cardiovascular system, such as atherosclerosis. Various members of the MMP family have been shown to be expressed in atherosclerotic lesions, but MMP9 is consistently seen in inflammatory atherosclerotic lesions. MMP9 over-expression is implicated in the vascular re-modelling events preceding plaque rupture (the most common cause of acute myocardial infarction). Reduced MMP9 activity, either by genetic manipulation or through pharmacological intervention, has an impact on ventricular re-modelling following infarction. MMP9 activity may therefore represent a key mechanism in the pathogenesis of heart failure. We have determined the crystal structure, at 2.3 A resolution, of the catalytic domain of human MMP9 bound to a peptidic reverse hydroxamate inhibitor as well as the complex of the same inhibitor bound to an active-site mutant (E402Q) at 2.1 A resolution. MMP9 adopts the typical MMP fold. The catalytic centre is composed of the active-site zinc ion, co-ordinated by three histidine residues (401, 405 and 411) and the essential glutamic acid residue (402). The main differences between the catalytic domains of various MMPs occur in the S1' subsite or selectivity pocket. The S1' specificity site in MMP9 is perhaps best described as a tunnel leading toward solvent, as in MMP2 and MMP13, as opposed to the smaller pocket found in fibroblast collagenase and matrilysin. The present structure enables us to aid the design of potent and specific inhibitors for this important cardiovascular disease target.

Role of inflammatory mediators in resistance and susceptibility to pneumococcal infection.

Variations in the host response during pneumonia caused by Streptococcus pneumoniae in susceptible (CBA/Ca) and resistant (BALB/c) inbred mouse strains were investigated. Significant differences were detected in survival time, core body temperature, lung-associated and systemic bacterial loads, mast cell numbers, magnitude and location of cytokine production, lung disruption, and ability of isolated lung cells to release the cytokine tumor necrosis factor (TNF) alpha in vitro. Overall, the results indicate that the reduced capacity of CBA/Ca mice to induce rapid TNF activity within the airways following infection with S. pneumoniae may be a factor in their elevated susceptibility to pneumococcal pneumonia.

Evaluation of the intranasal challenge route in mice as a mucosal model for Candida albicans infection.

The intranasal route was used to study Candida albicans infections in mice. Mice from two different inbred strains were challenged intranasally with C. albicans and the level of local and systemic colonization was monitored. DBA/2 mice were highly susceptible to challenge and viable C. albicans disseminated from the lungs to deeper tissues, including kidneys, liver and spleen within 48 h. In contrast, in BALB/c mice challenged in the same manner, C. albicans were retained within the lungs and cleared. Local and systemic anti-C. albicans immune responses were investigated. BALB/c mice exhibited higher titres of serum and mucosal anti-C. albicans IgA than DBA/2 mice. Splenocytes from BALB/c mice, but not from DBA/2 mice, produced detectable levels of interleukin-4 and -5 following stimulation with C. albicans antigens. Both DBA/2- and BALB/c-derived splenocytes produced interferon-gamma and interleukin-10 in response to similar stimulation. In conclusion, the intranasal route provided a simple, non-invasive murine model for investigating C. albicans infection through mucosal surfaces.